Process for production of dihomo-γ-linolenic acid and lipid containing same

ABSTRACT

A process for the production of dihomo-γ-linolenic acid comprising the steps of culturing a microorganism having an ability to produce araquidonic acid and having a reduced or lost Δ5 desaturase activity to produce dihomo-γ-linolenic acid or a lipid containing dihomo-γ-linolenic acid, and recovering the dihomo-γ-linolenic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/230,879, filed Apr. 20, 1994 (U.S. Pat. No. 6,280,982), which is aRule 62 Continuation of U.S. patent application Ser. No. 07/953,096,filed Sep. 29, 1992 (abandoned), incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for the production ofdihomo-γ-linolenic acid (DGLA) and a lipid containing DGLA byfermentation using a microorganism capable of producing arachidonic acid(ARA) and having reduced or lost Δ5 desaturation activity.

2. Related Art

DGLA, also known as 8,11,14-eicosatrienoic acid, is known to be presentas a constituent fatty acid of fish oils and seaweeds. However, becauseof the low content of DGLA in fish oils and the like, a purified DGLAproduct obtained from fish oils and seaweeds is expensive. As relativelyefficient production processes, there are fermentation methods using amicroorganism capable of producing DGLA (Japanese Unexamined PatentPublication No. 63-14696), methods providing enhanced productivity byadding an additive, such as an unsaturated fatty acid (JapaneseUnexamined Patent Publication Nos. 64-47384, and 64-47385), and methodsusing sesame oil, one of various plant extracts, sesamin, episesamin orthe like (Japanese Unexamined Patent Publication Nos. 1-243992,2-268690, 3-72892 and 3-49688).

Various studies have been carried out regarding the actions of essentialfatty acids on an organism, and it is known that in many caseseicosanoids derived from DGLA and ARA are antagonistic. Although it isknown that a group of prostaglandin 1 group derived from DGLA exhibitplatelet anti-coagulation action, vasodilation activity, bronchdilationactivity anti-inflammatory action, and the like, for DGLA orally takenas a fat or oil in foods so as to exhibit the above-mentioned actions,DGLA-containing oil or fat product that has a low content of ARAantagonistic to DGLA is most preferable. Moreover, in the case whereinDGLA is purified from a DGLA-containing fat or oil, a DGLA-containingfat or oil having a low content of ARA is preferable as a startingmaterial. Thus, the development of DGLA-containing fat or oil having alow content of ARA is in urgent demand, but a process for the productionof such a fat or oil is not known.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a process for the productionof DGLA and a lipid containing DGLA in a simple and efficient mannerusing a conventional inexpensive medium, and a process for theproduction of a lipid containing DGLA having a low content of ARA byadding an additive such as a Δ5 desaturase inhibitor.

The present invention have found that DGLA, which is a precursor of ARA,accumulates in a large amount when a microorganism capable of producingARA and having reduced or lost Δ5 desaturation activity is cultured in aconventional medium, and that a ratio of ARA is further decreased and aratio of DGLA is further increased when said microorganism is culturedin a medium supplemented with a Δ5 desaturation inhibitor.

Accordingly, the present invention provides a process for the productionof DGLA comprising the steps of:

culturing a microorganism having an ability to produce ARA and havingreduced or lost Δ5 desaturation activity in a medium to produce DGLA ora lipid containing DGLA; and

recovering the DGLA.

The present invention also provides a process for the production of alipid containing DGLA comprising the steps of:

culturing a microorganism having an ability to produce ARA and havingreduced or lost Δ5 desaturation activity in a medium to produce a lipidcontaining DGLA; and

recovering the lipid containing DGLA.

The present invention further provides a process for the production ofDGLA comprising the steps of:

culturing a microorganism having an ability to produce ARA and havingreduced or lost Δ5 desaturation activity in a medium supplemented with aΔ5 desaturase inhibitor, or adding a Δ5 desaturase inhibitor into amedium in which said microorganism has been cultured and furtherculturing the microorganism to produce DGLA or a lipid containing DGLA;and

recovering the DGLA.

The present invention further provides a process for the production of alipid containing DGLA comprising the steps of:

culturing a microorganism having an ability to produce ARA and havingreduced or lost Δ5 desaturation activity in a medium supplemented with aΔ5 desaturase inhibitor, or adding a Δ5 desaturase inhibitor into amedium in which said microorganism has been cultured and furtherculturing the microorganism to produce a lipid containing DGLA; and

recovering the lipid containing DGLA.

The present invention moreover provides a process for the production ofDGLA comprising the steps of:

culturing a microorganism having an ability to produce ARA and havingreduced or lost Δ5 desaturation activity in a medium supplemented withat least one additive selected from the group consisting of sesame oil,peanut oil, an extract obtained by extracting sesame oil with an organicsolvent substantially immiscible with sesame oil, an extract of sesameseeds, an extract of Gokahi derived from a medicinal plant, which isAcanthopanax gracilistylus W. W. Smith, Acanthopanax senticosus Harms,Acanthopanax henryi, or Acanthopanax verticillatus Hoo, an extract ofTouboku derived from a medicinal plant, an which is Paulownia fortuneiHemsl; or Paulownia tomentosa Steud., an extract of Hakukajihi derivedfrom a medicinal plant, an which is of Ginkgo biloba L., an extract ofHihatsu derived from a medicinal plant, an extract of Piper longum L.,which is Saishin (Asiasari radix) derived from medicinal plant, a whichis Asiasarum heterotropoides var. mandshuricum, an extract of Asarumsieboldii Miq., an extract of tarragon, an extract of dill seeds, anextract of parsley, an extract of turmeric and an extract of nutmeg, oradding said additive into a medium in which said microorganism has beencultured and further culturing the microorganism to produce DGLA or alipid containing DGLA, and

recovering the DGLA.

The present invention further provides a process for the production of alipid containing DGLA comprising the steps of:

culturing a microorganism having an ability to produce ARA and havingreduced or lost Δ5 desaturation activity in a medium supplemented withat least one additive selected from the group consisting of sesame oil,peanut oil, an extract obtained by extracting sesame oil with an organicsolvent substantially immiscible with sesame oil, an extract of sesameseeds, an extract of Gokahi derived from a medicinal plant, an extractof Acanthopanax gracilistylus W. W. Smith, an extract of Acanthopanaxsenticosus Harms, an extract of Acanthopanax henryi, an extract ofAcanthopanax verticillatus Hoo, an extract of Touboku derived from amedicinal plant, an extract of Paulownia fortunei Hemsl; an extract ofPaulownia tomentosa Steud., an extract of Hakukajihi derived from amedicinal plant, an extract of Ginkgo biloba L., an extract of Hihatsuderived from a medicinal plant, an extract of Piper longum L., anextract of Saishin (Asiasari radix) derived from medicinal plant, anextract of Asiasarum heterotropoides var. mandshuricum, an extract ofAsarum sieboldii Miq., an extract of tarragon, an extract of dill seeds,an extract of parsley, an extract of turmeric and an extract of nutmeg,or adding said additive into a medium in which said microorganism hasbeen cultured and further culturing the microorganism to produce a lipidcontaining DGLA, and

recovering the lipid containing DGLA.

DETAILED DESCRIPTION

In the present invention any microorganisms having an ability to produceARA and having reduced or lost Δ5 desaturase activity can be used.Microorganism having an ability to produce ARA include those belongingto the genus Mortierella, Conidiobolus, Pythium, Phytophthora,Penicillium, Cladosporium, Mucor, Fusarium, Aspergillus, Rhodotorula, orEntomophthora. As microorganisms belonging to the genus Mortierella,there are mentioned microorganisms belonging to the subgenusMortierella, such as Mortierella elongata, Mortierella exiqua,Mortierella hyprophila, Mortierella alpina, and the like. Microorganismused in the present invention, having an ability to produce ARA andhaving reduced or lost Δ5 desaturase activity can be obtained bymutating the microorganisms having an ability to produce ARA.

For mutagenesis, irradiation of a microorganism with a mutagen, such asradiation (X-ray, γ-ray, neutron or ultraviolet light), high temperaturetreatment, and chemical mutagens may be used. In a mutagenizingprocedure, microbial cells are suspended to an appropriate buffer, and amutagen is added therein. The treated suspension is incubated for anappropriate time, diluted and plated on a solid medium such as agarmedium to form colonies of mutated microorganisms.

As chemical mutagens, alkylating agents such as nitrogen mustard, methylmethanesulfonate (MMS), N-methyl-N′-nitro-N-nitrosoguanidine (NTG); baseanalogs such as 5-bromouracil; antibiotics such as mitomycin C; basesynthesis inhibitor such as 6-mercaptopurine; pigments such asproflavine; a certain carcinogens such as 4-nitroquinoline-N-oxide; andothers such as manganese chloride, potassium permanganese, nitrous acid,hydrazine, hydroxylamine, formaldehyde, nitrofurane compounds may bementioned. Microorganisms to be treated with a mutagen can be vegetativecells such as mycelium or spores.

As a mutant belonging to the genus Mortierella, Mortierella alpina SAM1860 (FERM BP-3589) can be preferably used.

For culturing a mutant used in the present invention, spores, myceliumor a previously cultured precultur is inoculated to a liquid medium orsolid medium. A liquid medium contains, as a carbon source, glucose,fructose, xylose, saccharose, maltose, soluble starch, molasses,glycerol, mannitol, and the like, alone or in combination.

As a nitrogen source, an organic nitrogen source such as peptone, yeastextract, malt extract, meat extract, casamino acids, corn steep liquoror urea, and an inorganic nitrogen source such as sodium nitrate,ammonium nitrate, ammonium sulfate or the like can be used alone or incombination. In addition, if necessary, inorganic salts such asphosphates, magnesium sulfate, ferric or ferrous sulfate, cupric sulfateor the like, and minor nutrient components such as vitamins may be used.

The concentration of components in a culture medium should be such thatit does not inhibit the growth of microorganisms. Generally andpractically, a concentration of carbon source is 0.1 to 30% by weight,preferably 1 to 10% by weight, and a concentration of nitrogen source is0.01 to 5% by weight, and preferably 0.1 to 2% by weight. Temperaturefor culturing is 5 to 40° C., and preferably 20 to 30° C.; and a pHvalue of a medium is 4 to 10, and preferably 6 to 9. Culturing may beaeration/agitation culturing, shaking culture, or stationary culture.Culturing is usually continued for 2 to 10 days.

In the case wherein a microorganism is cultured in a solid medium, themedium comprises wheat bran, rice hulls, rice bran or the likesupplemented with water in an amount of 50 to 100% by weight retuting tothe weight of solid materials. Culturing is carried out at 5 to 40° C.,preferably 20 to 30° C. for 3 to 14 days. In this case, the medium cancontain nitrogen sources, inorganic salts, and minor nutrient compounds,such as those described above.

According to the present invention, to accelerate an accumulation ofDGLA, a substrate of ARA can be added to a medium. As the substrates,hydrocarbons having 12 to 20 carbon atoms such as tetradecane,hexadecane and octadecane, fatty acid having 12 to 20 carbon atoms suchas tetradecanoic acid, hexadecanoic acid and octodecanoic acid, a saltsthereof, for example, sodium salt or potassium salt, fatty acid esterswherein the fatty acid moiety has 12 to 20 carbon atoms, for example,lower alkyl ester, for example, methyl ester, ethyl ester or propylester of such a fatty acid, and a lipid containing such fatty acids asits components, for example, olive oil, soybean oil, cotton seed oil,coconut oil may be mentioned, and they can be used alone or incombination.

Moreover, according to the present invention, to produce a lipidcontaining DGLA in which a content of ARA is decreased, a producermicroorganism is cultured in the presence of a Δ5 desaturase inhibitor,resulting in the accumulation of a large amount of DGLA. In this case,as Δ5 desaturase inhibitors, there are mentioneddioxabicyclo[3.3.0]octane derivatives represented by the followingformula (I):

wherein R¹, R², R³, R⁴, R⁵ and R⁶ independently represent a hydrogenatom or an alkyl group having 1 to 3 carbon atoms, or R¹ and R², and/orR⁴ and R⁵ together form a methylene group or an ethylene group, and n, mand l are 0 or 1;

piperonyl butoxide, curcumin, and compounds represented by the followingformula (II):

wherein R¹ represents a lower alkyl group; R² represents a hydroxylgroup, an alkyl group, an alkoxy group, an alkenyl group or an oxyalkylgroup wherein in the case that more than one R² is present, the R² maybe the same or different, and n is an integer of 0 to 5. The lower alkylgroup has 1 to 6 carbon atoms, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl and the like. The alkyl group,the alkyl moiety in the alkoxy group, or the oxyalkyl group, and thealkenyl group have 12 to 20 carbon atoms. The Δ5 desaturase inhibitorscan be used alone or in combination.

As the dioxabicyclo[3.3.0]octane derivative, in the present invention,sesamin, sesaminol, episesamin, episesaminol, sesamolin,2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo-[3.3.0]octane,2,6-bis-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane or2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]octane,for example, can be used. These derivatives can be used alone or in theform of a mixture of two or more thereof. Both the optically active formand racemic form can be used.

The dioxabicyclo[3.3.0]octane derivative, one of the Δ5 desaturaseinhibitors of the present invention can be obtained by the followingprocedure. First, an extract composed mainly of thedioxabicyclo[3.3.0]octane derivatives can be obtained from sesame oilaccording to a method comprising extracting sesame oil with an organicsolvent substantially immiscible with sesame oil and capable ofextracting and dissolving the compound of the present invention, andconcentrating the extract. As the organic solvent, there can bementioned, for example, acetone, methylethylketone, diethylketone,methanol and ethanol. For example, an extract composed mainly of thecompound of the present invention can be obtained by mixing sesame oilhomogeneously with an organic solvent as mentioned above, allowing themixture to stand at a low temperature, carrying out a phase separationaccording to a customary process, and removing the solvent from thesolvent fraction by evaporation.

More specifically, sesame oil is dissolved in 2 to 10 volumes,preferably 6 to 8 volumes of acetone, and the solution is allowed tostand at −80° C. overnight. As a result, the oil component isprecipitated, and the organic solvent is removed from the obtainedfiltrate by distillation, whereby an extract composed mainly of thecompound of the present invention is obtained. Alternatively, sesame oilis mixed with hot methanol or hot ethanol, the mixture is allowed tostand at room temperature, and the solvent is removed from the solventfraction to obtain an extract composed mainly of the compound of thepresent invention. More specifically, sesame oil is mixed with hotmethanol (higher than 50° C.) or hot ethanol (higher than 50° C.) in avolume 2 to 10 times, preferably 5 to 7 times, as large as the volume ofthe sesame oil to effect a violent extraction. The phase separation iseffected by a phase separation when standing at room temperature or acentrifugal separation according to customary procedures, and thesolvent is removed from the solvent fraction by distillation to obtainan extract composed mainly of the compound used in the presentinvention. Furthermore, the supercritical gas extraction can beutilized.

The compound of the present invention can be obtained from an extract asmentioned above by treating the extract by a customary method such ascolumn chromatography, high performance liquid chromatography,recrystallization, distillation, or liquid—liquid countercurrentdistribution chromatography. More specifically, by using a reversedphase column (5C₁₈) and methanol/water (60/40) as the eluent, theextract is subjected to high performance liquid chromatography, thesolvent is removed by distillation, and the obtained crystal isrecrystallized from ethanol to obtain the compound used in the presentinvention, such as sesamin, episesamin, sesaminol or episesaminol. Thesesame oil used in the present invention can be either a purifiedproduct or a crude product. Furthermore, sesame seeds or sesame lees(defatted sesame seeds having a residual oil content of 8 to 10%) can beused. In this case, sesame seeds or sesame lees are pulverized ifnecessary, and then subjected to the extraction according to customaryprocedures using any solvent, for example, a solvent as mentioned abovewith respect to the extraction from sesame oil. The extraction residueis separated, and the solvent is removed from the extract by evaporationor the like to obtain an extraction product.

The compound used in the present invention, for example, sesamin,sesaminol, episesamin, episesaminol, sesamolin,2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane,2,6-bis-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane or2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]octane,can be obtained from a sesame seed extract, a sesame lee extract or acrude sesame oil extract according to the same procedures as describedabove. Moreover, the compound used in the present invention can beobtained from a by-product formed in the sesame oil-preparing process.

Note, sesamin obtained from Asaiasari radix exhibits the same effects asthose provided by sesame seeds, sesame bran and sesame oil.

The process for the purification of the compound used in the presentinvention and the process for obtaining the extract are not limited tothose mentioned above, and the compound used in the present inventionand the extract composed mainly of the compound of the present inventionare not limited to those obtained from sesame oil, sesame lees andsesame seeds, but as is apparent to persons with ordinary skill in theart, all natural substances containing the compound used in the presentinvention can be used. For example, there can be mentioned Gokahiderived from a medicinal plant which is Acanthopanax gracilistylus W. W.Smith, Acanthopanax senticosus Harms, Acanthopanax henryior,Acanthopanax verticillatus Hoo, Touboku derived from a medicinal plantwhich is Paulownia fortunei Hemslor, Paulownia tomentosa Steud.,Hakukajihi derived from a medicinal plant which is Ginkgo biloba L.,Hihatsu derived from a medicinal plant which is Piper longum L., Saishin(Asiasari radix) derived from medicinal plant which is Asiasarumheterotropoides var. mandshuricumor Asarum sieboldii Miq.

The following processes can be adopted for the synthesis of thedioxabicyclo[3.3.0]octane derivative.

For example, sesamin and episesamin can be synthesized according to theprocess of Beroza et al. [J. Am. Chem. Soc., 78, 1242 (1956)].Pinoresinol [in the general formula (I), R¹ and R⁴ represent H, R² andR⁵ represent CH₃, and n, m and l are zero] can be synthesized accordingto the process of Freundenberg et al. [Chem. Ber., 86, 1157 (1953)].Furthermore, syringaresinol [in the general formula (I), R¹ and R⁴represent H, R², R³, R⁵ and R⁶ represent CH₃, n is zero, and each of mand l is 1] can be synthesized according to the process of Freundenberget al. [Chem. Ber., 88, 16 (1955)].

The compound used in the present invention also can be used in the formof a glycoside, to accelerate absorption as far as the glycoside has aspecific Δ⁵ desaturase inhibitory activity.

As embodiments of the compound represented by the formula (II), anisole,methoxyphenol, dimethoxybenzene, diethoxybenzene, trimethoxybenzene,methoxytoluene, 3(2)-tert-butyl-4-hydroxanisole (BHA), eugenol, and thelike can be mentioned.

Moreover, as additives added to a culture medium to increase anaccumulation of DGLA, sesame oil, peanut oil, an extract obtained byextracting sesame oil with an organic solvent substantially immisciblein sesame oil, an extract of sesame seeds, an extract of Gokahi derivedfrom a medicinal plant, an extract of Acanthopanax gracilistylus W. W.Smith, an extract of Acanthopanax senticosus Harms, an extract ofAcanthopanax henryi, an extract of Acanthopanax verticillatus Hoo, anextract of Touboku derived from a medicinal plant, an extract ofPaulownia fortunei Hemsl; an extract of Paulownia tomentosa Steud., anextract of Hakukajihi derived from a medicinal plant, an extract ofGinkgo biloba L., an extract of Hihatsu derived from a medicinal plant,an extract of Piper longum L., an extract of Saishin (Asiasari radix)derived from medicinal plant, an extract of Asiasarum heterotropoidesvar. mandshuricum, an extract of Asarum sieboldii Miq., as well asextracts of spicy plants, such as an extract of tarragon, an extract ofdill seeds, an extract of parsley, an extract of turmeric, an extract ofnutmeg and the like can be used. These extracts can be prepared using asolvent such as dichloromethane, ethanol, methanol, ethyl ether or thelike.

An amount of the above-mentioned additives to be added to a culturemedium is as follow. An amount of sesame oil or peanut oil or a totalamount of them is 0.001 to 10% by weight per medium, and preferably 0.5to 10% by weight per medium. An amount of a sesame oil extract and otherextract to be added is 3×10⁻³ to 3×10⁻¹% by weight per medium. An amountof a dioxabicyclo[3.3.0]octane derivatives such as sesamin, sesaminol,episesamin, episesaminol and the like, or a total amount of acombination thereof is 1×10⁻³ to 1×10⁻¹% by weight per medium.

The additive can be added prior to the inoculation of a producermicroorganism or immediately after the inoculation. Alternatively, theadditive can be added, after the culturing has started, to a culturemedium in which the microorganism is growing or has been grown, followedby further culturing. Moreover, the additive can be added both prior toculturing and during culturing after the culturing has started. In thecase wherein the additive is added during culturing, the additive can beadded once or more than one time, or continuously.

During the culturing, a large amount of lipid containing DGLA isintracellularly accumulated. In the case wherein a liquid medium isused, DGLA is then recovered by a procedure, for example, described inthe following.

After the culturing, the cultured cells are recovered by a conventionalsolid liquid separation means, such as centrifugation or filtering. Thecells are thoroughly washed with water, and preferably dried. The dryingcan be carried out by lyophilization or air drying. The dried cells areextracted with an organic solvent, preferably in a nitrogen gas flow. Asan organic solvent, an ether such as ethyl ether, hexane, a loweralcohol such as methanol or ethanol, chloroform, dichloromethane,petroleum ether, or the like can be used. Moreover, an alternatingextraction with methanol and petroleum ether, or an extraction with onephase solvent of chloroform-methanol-water can be successfully used. Thesolvent is distilled off from the extract under reduced pressure toobtain a lipid containing DGLA in a high concentration.

Alternatively, wet cells can be extracted with a solvent miscible withwater, such as methanol or ethanol, or a mixed solvent miscible withwater, comprising said solvent and water and/or other solvent. Otherprocedures are the same as described above for dried cells.

The lipid thus obtained contains DGLA as a component of the lipid suchas fat. Although DGLA can be directly isolated, preferably it isisolated as an ester with a lower alcohol, for example, as methyldihomo-γ-linolenate. The esterification accelerates the separation ofthe target fatty acid from other lipid components, and from other fattyacids produced during the culturing, such as palmitic acid, oleic acidand linoleic acid (these fatty acids are also esterified simultaneouslywith the esterification of DGLA). For example, to obtain methyl ester ofDGLA, the above-mentioned extract is treated with anhydrous methanol/HCl5 to 10%, or BF₃/methanol 10 to 50% at room temperature for 1 to 24hours.

Methyl ester of DGLA is recovered preferably by extracting theabove-mentioned treated solution with an organic solvent such as hexane,an ether such as ethyl ether, or an ester such as ethyl acetate. Next,the resulting extract is dried on, for example, anhydrous sodiumsulfate, and the solvent is distilled off preferably under reducedpressure to obtain a mixture comprising fatty acid esters. This mixturecontains, in addition to the desired fatty acid HGLA methyl ester, otherfatty acid methyl esters, such as methyl parmitate, methyl stearate,methyl oleate and the like. To isolate methyl ester of DGLA from themixture of these fatty acid methyl esters, column chromatography, lowtemperature crystallization, the urea-inclusion method, theliquid/liquid countercurrent chromatography method, and the like can beused alone or in combination.

To obtain DGLA from the methyl ester of DGLA, the latter is hydrolyzedwith an alkali and freed DGLA is then extracted with an organic solvent,for example, an ether such as an ethyl ether, an ester such as ethylacetate, or the like.

Moreover, to recover DGLA without using methyl ester, theabove-mentioned extracted lipid is subjected to an alkalysis (forexample, with 5% sodium hydroxide at room temperature for 2 to 3 hours),and the alkali hydrolysate is extracted and the desired fatty acid DGLAis purified according to a conventional procedure.

EXAMPLES

Next, the present invention is further explained by Examples.

Example 1

100 ml of a medium (pH 6.0) containing 2% glucose and 1% yeast extractwas put into a 500 ml Erlenmeyer flask, which was then autoclaved at120° C. for 20 minutes. Mortierella alpina IFO 8568 (ComparativeExample) and a mutant SMA 1860 (Example) were separately added to themedia, and cultured on a reciprocating shaker (110 rpm) at 28° C. for 6days. After the culturing the cultured cells were recovered byfiltering, washed with water, and lyophilizated to obtain 1.28 g ofdried cells from the strain IFO 8568, and 1.37 g of dried cells from themutant SAM 1860.

These cells were extracted with a solvent of one phasechloroform/methanol/water according to Bligh & Dyer extraction method toobtain total lipids in an amount of 505 mg from the strain IFO 8568, andan amount of 530 mg from the mutant SAM 1860. These lipids weresubjected to methyl-esterification using anhydrous methanol/hydrochloricacid (95:5) at 50° C. for 3 hours, and the resulting fatty acid methylesters were extracted to obtain 417 mg of a total fatty acid methylester preparation from the strain IFO 8568, and 454 mg of a total fattyacid preparation from the mutant SAM 1860. The fatty acid methyl esterpreparations were analyzed by gas chromatography. The result is shown inTable 1.

TABLE 1 Strains Mortierella alpina Mutant Fatty acid IFO 8568 SAM 1860Palmitic acid 0.79 0.77 Oleic acid 0.76 0.74 DGLA 0.20 1.27 ARA 1.420.46 Total fatty acid 3.95 4.30 Dried cells (g/L) 12.8 13.7

The amount of fatty acid is shown in grams per liter medium.

As seen from Table 1, the mutant SAM 1860, whose Δ5 desaturase activitythat converts DGLA to ARA is remarkably lowered in comparison with itsparent strain, accumulated a large amount of DGLA which is a precursorof ARA. Note, the fatty acid methyl esters were separated by columnchromatography to obtain a DGLA methyl ester fraction, which was thenanalyzed by gas chromatography, high performance liquid chromatography,mass spectrum and NMR analysis. As a result the data conformed to thoseobtained for a commercial preparation.

Example 2

100 ml of a medium (pH 6.0) containing 2% glucose and 1% yeast extract,a medium (pH 6.0) containing 4% glucose and 1% yeast extract, and amedium (pH 6.0) containing 8% glucose and 1% yeast extract were put intodifferent 500 ml Erlenmeyer flasks, and autoclaved at 120° C. for 20minutes.

A mutant Mortierella alpina SAM 1860 was added to the media, andcultured on a reciprocating shaker (110 rpm) at 28° C. or 20° C. for 10days. After the culturing, fatty acid methyl ester preparations preparedaccording to the same procedure as described in Example 1 were analyzedby gas chromatography. The result is shown in Table 2.

TABLE 2 Yield/medium Culture Glucose Dry cells (g/L) temperatureConcentration (g/L) DGLA ARA 28° C. 2% 11.0 1.33 0.49 4% 15.5 2.49 0.998% 20.5 3.72 1.27 20° C. 2% 11.8 1.51 0.50 4% 16.4 3.22 1.02 8% 20.04.10 1.20

As seen from Table 2, an increase of glucose increased an amount ofaccumulated DGLA, which is a precursor of ARA. On the other hand, in anycase an amount of ARA was suppressed to about 30 to 40% of DGLA. Theproductivity of DGLA was good at both culture temperatures of 28° C. and20° C.

Example 3

20 ml of a medium (pH 6.0) containing 2% glucose; 1% yeast extract; 0.2%Tween 20; and 0.5% hydrocarbon, sodium salt of fatty acid, fatty acidester, or fat or oil, was put into a 100 ml Erlenmeyer flask, andautoclaved at 120° C. for 20 minutes.

A mutant Mortierella alpina SAM 1860 was added to each flask, andcultured on a reciprocating shaker (110 rpm) at 28° C. for 7 days. Forthe resulting cell preparations, fatty acid methyl esters were analyzedby gas chromatography as described in Example 1. The result is shown inTable 3.

TABLE 3 Dry cells DGLA ARA Additives (g/L) (g/L) (g/L) Hexadecane 18.11.89 0.55 Octadecane 18.3 1.80 0.50 Sodium oleate 15.5 1.40 0.48 Sodiumlinoleate 14.8 1.40 0.49 Methyl oleate 18.1 1.99 0.56 Methyl linoleate18.7 2.15 0.60 Olive oil 17.9 1.92 0.59 Cotton seed oil 18.5 1.98 0.59Coconut oil 18.4 1.88 0.58 None additive 13.0 1.25 0.40

When hydrocarbon, sodium salt of fatty acid or oil is added to a basalmedium, productivity of DGLA increased by 12 to 72%.

Example 4

5 liters of medium (pH 6.0) containing 2% glucose, 1% yeast extract and0.1% soybean oil was put into a 5 liter jar fermenter, and aftersterilizing at 120° C. for 40 minutes, 200 ml of preculture of a mutantMortierella alpina SAM 1860 was inoculated therein. Culturing wascarried out at 28° C. and with aeration at 0.5 v.v.m and agitation for 7days, and during culturing 150 ml each of 33% glucose solution was addedon the second, third, fourth and fifth days.

960 g (dry weight 126 g) of wet cells thus obtained were treated asdescribed in Example 1 to obtain 74.8 g of total lipid and 70.2 g of afatty acid methyl ester mixture. DGLA content was 30% relating to totalfatty acid content, and DGLA productivity was 4.0 g/l, and 165 mg/g drycells.

Example 5

100 ml each of a medium (pH 6.0) containing 4% glucose and 1% yeastextract was put into 4 Erlenmeyer flasks having a volume of 500 ml, and0.5 ml each of soybean oil in which sesamin(2,6-bis-(3,4-methylenedioxyphenyl)-cis-3,7-dioxabicyclo[3,3,0]octane)had been dissolved to a sesamin concentration of 15% by weight was addedto 2 flasks, and 0.5 ml each of soybean oil was added 2 other flasks,and the flasks were sterilized at 120° C. for 20 minutes.

Mortierella alpina IFO 8568 (Comparative Example) was added to twodifferent media, and the mutant SAM 1860 (Example) was add to otherdifferent media.

The strains were cultured on a reciprocating shaker (110 rpm) at 28° C.for 7 days. After 24 hours from the start of culturing, 0.5 ml each ofthe sesamin-containing soybean oil was added to the media to which thesesamin-containing soybean oil had been added, and 0.5 ml each ofsoybean oil was added to the media to which soybean oil had been added.After culturing, a fatty acid methyl ester preparation was analyzed bygas chromatography as described in Example 1. The result is shown inTable 4.

TABLE 4 Strains M. alpina Mutant Addition of sesamin- IFO 8568 SAM 1860containing soybean oil − + − + Ratio of DGLA:ARA 0.15 1.17 5.04 12.3Total fatty acid (g/L) 11.1 11.8 12.0 11.9 Dry cells (g/L) 23.8 24.224.6 24.1 DGLA Productivity (g/L) 0.39 1.87 2.45 2.93

As seen from Table 4, in culturing the strain IFO 8568, even thoughsesamin which is a Δ5 desaturase inhibitor was added, the ratio ofDGLA:ARA did not increase higher than 1.17. On the other hand, inculturing the mutant SAM 1860 whose Δ5 desaturase activity had beendecreased, the addition of a Δ5 desaturase inhibitor, sesamin, increasedthe ratio of DGLA:ARA to 12.3. Note, the addition of sesamin did noteffect the amount of total fatty acids produced and the amount of drycells.

As seen from the above-mentioned results, since it is clear that theproduction of ARA is suppressed and the production of DGLA is increased,and a ratio of DGLA:ARA is increased by the same effects as described inJapanese Unexamined Patent Publication No 1-243992, then it is clearthat besides sesamin, other additives, such as sesame oil, peanut oil,an extract obtained by extracting sesame oil with an organic solventsubstantially immissible with sesame oil, an extract of sesame seeds,sesamin, sesaminol, episesamin, episesaminol, sesamolin,2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-(3,7-dioxabicyclo[3.3.0]octane,2,6-bis-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane,2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-(3,7-dioxabicyclo[3.3.0]octane,an extract of Gokahi derived from a medicinal plant, an extract ofAcanthopanax gracilistylus W. W. Smith, an extract of Acanthopanaxsenticosus Harms, an extract of Acanthopanax henryi, an extract ofAcanthopanax verticillatus Hoo, an extract of Touboku derived from amedicinal plant, an extract of Paulownia fortunei Hemsl; an extract ofPaulownia tomentosa Steud., an extract of Hakukajihi derived from amedicinal plant, an extract of Ginkgo biloba L., an extract of Hihatsuderived from a medicinal plant, an extract of Piper longum L., anextract of Saishin (Asiasari radix) derived from medicinal plant, anextract of Asiasarum heterotropoides var. mandshuricum, an extract ofAsarum sieboldii Miq., as well as extracts of plants, such as an extractof tarragon, an extract of dill seeds, an extract of parsley, an extractof turmeric, and an extract of nutmeg suppress the production of ARA andincrease the production of DGLA, and increase the ratio of DGLA:ARA.

What is claimed is:
 1. A dihomo-γ-linolenic acid-containing lipidwherein the ratio of dihomo-γ-linolenic acid per arachidonic acid in thelipid is at least 12.3 parts by weight.
 2. A dihomo-γ-linolenicacid-containing microbial lipid, containing at least 30% by weightdihomo-γ-linolenic acid per total fatty acids in the lipid.
 3. Adihomo-γ-linolenic acid-containing lipid which containsdihomo-γ-linolenic acid as a component of fat, wherein the ratio ofdihomo-γ-linolenic acid per arachidonic acid in the lipid is at least12.3 parts by weight.
 4. A dihomo-γ-linolenic acid-containing microbiallipid, wherein the ratio of dihomo-γ-linolenic acid per arachidonic acidin the lipid is at least 12.3 parts by weight.